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Local administration of calcitriol positively influences bone remodeling andmaturation during restoration of mandibular bone defects in rats

Hongrui Liu, Jian Cui, Wei Feng, Shengyu Lv, Juan Du, Jing Sun, Xi-uchun Han, Zhenming Wang, Xiong Lu, Yimin, Kimimitsu Oda, NorioAmizuka, Minqi Li

PII: S0928-4931(14)00862-5DOI: doi: 10.1016/j.msec.2014.12.064Reference: MSC 5149

To appear in: Materials Science & Engineering C

Received date: 10 July 2014Revised date: 26 November 2014Accepted date: 17 December 2014

Please cite this article as: Hongrui Liu, Jian Cui, Wei Feng, Shengyu Lv, Juan Du, JingSun, Xiuchun Han, Zhenming Wang, Xiong Lu, Yimin, Kimimitsu Oda, Norio Amizuka,Minqi Li, Local administration of calcitriol positively influences bone remodeling andmaturation during restoration of mandibular bone defects in rats, Materials Science &Engineering C (2014), doi: 10.1016/j.msec.2014.12.064

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Local administration of calcitriol positively influences bone remodeling and

maturation during restoration of mandibular bone defects in rats

Hongrui Liu§1

, Jian Cui§1

, Wei Feng1, Shengyu Lv

1, Juan Du

1, Jing Sun

1, Xiuchun

Han1, Zhenming Wang

2, Xiong Lu

2, Yimin

3, Kimimitsu Oda

4, Norio Amizuka

5, Minqi

Li1

1Department of Bone Metabolism, School of Stomatology Shandong University,

Shandong Provincial Key Laboratory of Oral Biomedicine, Jinan, China 2Key Lab of

Advanced Technologies of Materials, Ministry of Education, School of Materials

Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan, China 3

Department of Advanced Medicine, Graduate School of Medicine, Hokkaido

University, Sapporo, Japan 4Division of Biochemistry, Niigata University Graduate

School of Medical and Dental Sciences, Niigata, Japan and 5Department of

Developmental Biology of Hard Tissue, Graduate School of Dental Medicine,

Hokkaido University, Sapporo, Japan

Address for correspondence:

Minqi Li, DDS, PhD

Department of Bone Metabolism

School of Stomatology Shandong University

Shandong Provincial Key Laboratory of Oral Biomedicine

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Wenhua West Road 44-1, Jinan 250012, China

Tel & Fax: +86-531-88382095

E-mail: liminqi@sdu.edu.cn

§ We regard these authors equally contributed to this article.

Abstract

The aim of this study was to investigate the influence of calcitriol on osteoinduction

following local administration into mandibular bone defects. Calcitriol-loaded

absorbable collagen membrane scaffolds were prepared using the polydopamine

coating method and characterized by scanning electron microscopy. Composite

scaffolds were implanted into rat mandibular bone defects in the following groups: no

graft material (control), bare collagen membrane (CM group), collagen membrane

bearing polydopamine coating (DOP/CM group), and collagen membrane bearing

polydopamine coating absorbed with calcitriol (CAL/DOP/CM group). At 1, 2, 4 and

8 weeks post-surgery, the osteogenic potential of calcitriol was examined by

histological and immunohistochemical methods. Following in vivo implantation,

calcitriol-loaded composite scaffolds underwent rapid degradation with pronounced

replacement by new bone and induced reunion of the bone marrow cavity. Calcitriol

showed strong potential in inhibiting osteoclastogenesis and promotion of osteogenic

differentiation at weeks 1, 2. Furthermore, statistical analysis revealed that the newly

formed bone volume in the CAL/DOP/CM group was significantly higher than other

groups at weeks 1, 2. At weeks 4, 8, the CAL/DOP/CM group showed more

mineralized bone and uniform collagen structure. These data suggest that local

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administration of calcitriol is promising in promoting osteogenesis and mineralization

for restoration of mandibular bone defects.

Keywords: calcitriol, osteoclast, osteoblast, mineralization, mandibular bone defect

Running title: Calcitriol promotes repair of mandibular bone defects

Introduction

Achieving satisfactory bone regeneration in vivo remains an important goal in

orthopedic and dental clinical applications. Growth-factor-based tissue engineering

technologies have been widely studied, with much of this research focused on bone

morphogenetic proteins (BMPs), which have favorable osteogenic potential (Han et al.

2014; Muzio et al. 2014; Sigurdsson et al. 2001; Tatakis et al. 2002; Wang et al. 2014).

Despite that, their use is going to be limited due to several drawbacks including their

rapid degradation, high costs, safety and efficacy concerns, osteolysis, ectopic bone

formation, and soft tissue swelling (Arrabal et al. 2013; Ehnert et al. 2012; Oryan et al.

2014). Recently, it was reported that some drugs and bioactive substances which have

stable and srong activity, low costs and high biological safety could also be used to

regulate tissue growth (Jang et al. 2003; Kim et al. 2006). Calcitriol, also called

1,25-dihydroxyvitamin D3 (1, 25(OH)2D3), is the active form of vitamin D3 and is

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involved in various physiological processes, including calcium homeostasis, bone

metabolism and immune response (Inada et al. 2008). It functions by binding to a

single vitamin D receptor (VDR), which is a member of the nuclear hormone receptor

superfamily (Feldman et al. 2005). Vitamin D-deficiency causes impairment of bone

mineralization, resulting in rickets in infants and osteomalacia in adults (Suda et al.

2003). Moreover, mice lacking VDR exhibit impaired bone formation (Yoshizawa et

al. 1997). Conversely, administration of vitamin D to ovariectomized or rachitic

animals can relieve impaired bone mineralization (Shiraishi et al. 2002). In addition,

clinical studies show that vitamin D treatment can reduce bone fracture risk in

age-related bone loss, postmenopausal osteoporosis and drug-induced osteoporosis

(Avenell et al. 2005; de Nijs et al. 2004; Nawata et al. 2005). Besides the overall

positive effects on bone, calcitriol has also been shown to regulate transcription of the

collagen gene in osteoblasts (Harrison et al. 1989) and is a potent transcriptional

activator of genes encoding alkaline phosphatase (ALP), osteocalcin (OCN) and

osteopontin (OPN) (Noda et al. 1990). Moreover, osteoblasts have shown increased

activity after 1, 25(OH)2D3 treatment in vitro (Bosetti et al. 2007).

Based on these studies, we aimed to investigate local administration of calcitriol

for the restoration of mandibular bone defects in rats. However, the lack of a suitable

local delivery system has previously impeded in vivo application of calcitriol. An

appropriate scaffold with good biocompatibility and biodegradability is therefore

required. More importantly, an optimal amount of calcitriol to generate osteoinduction

needs to be released into the target site over a prolonged period of time. Absorbable

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collagen membrane, a well-documented barrier membrane in guided bone

regeneration (GBR) (Chiapasco and Zaniboni 2009; Hämmerle et al. 2002), has been

used as a growth-factor-combined scaffold. Type-I collagen, as the main component

of collagen membrane, is a natural and ubiquitous protein. Its inherent properties

include cell attachment and bioactivity, natural biodegradability, amenability to

chemical modifications, mechanical cross-linking and self-assembly into a fibrillar gel

under physiological conditions, making collagen advantageous for tissue engineering

and regenerative medicine applications (Drzewiecki et al. 2014). In addition, it also

showed significant strength and rigidity which can be attributed to its three

polypeptide chains structure (Drzewiecki et al. 2014). Numerous animal studies have

shown that collagen membrane is crucially important in retaining BMP levels and

ensuring maintenance of space for new bone formation (Khan et al. 2012). Moreover,

it is reported that loading of collagen membrane with rhBMP-2 shows favorable bone

regenerative ability (Jung et al. 2003). In view of these results, we selected collagen

membrane as the scaffold for loading calcitriol. However, to achieve enhanced

immobilization of calcitriol, this carrier material requires additional processing.

Various techniques are available for immobilizing biomolecules onto scaffolds,

including gamma ray irradiation and plasma treatment (Cottam et al. 2009; Nagai et al.

2006; Vallières et al. 2007; Xia et al. 2010). However, these methods have limitations

in terms of penetration depth and possible cleavage of polymeric chains (Nyström et

al. 2010). Recently, a versatile surface modification method, by simply dip-coating

with dopamine solution, was reported, and can be applied to almost all solid materials

from metals to synthetic polymers (Lee et al. 2007). Dopamine can undergo oxidative

polymerization and form a stable layer adherent to the surface of biomaterials under

basic conditions. Previous studies have applied the polydopamine-coated method to

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immobilizing animated methoxy-polyethylene glycol, trypsin, bovine serum albumin

(BSA), and antibodies (Lynge et al. 2011). In addition, it is reported that growth

factors, such as vascular endothelial growth factor and BMP-2, immobilized onto

titanium surfaces following polydopamine coating, promote proliferation of

endothelial cells and differentiation of rat mesenchymal stem cells, respectively

(Lynge et al. 2011). Most importantly, polydopamine has shown excellent

biocompatibility and low cytotoxicity, making it a favorable platform (Cui et al. 2012;

Yang et al. 2012).

In this study, we loaded calcitriol onto collagen membranes using the

polydopamine-coating method and evaluated the osteogenic capability of calcitriol by

applying the composite scaffold into mandibular bone defects in rats.

Materials and Methods

Immobilization of calcitriol on collagen membrane and SEM observation

Dopamine solution (2mg/ml) was prepared by dissolving 3,4-dihydroxyphenylamine

(Sigma-Aldrich, St. Louis, MO, USA) in Tris-HCl buffer (10 mM, pH 8.5), in

accordance with a previous study (Yang et al. 2012). Calcitriol (Selleck Chemicals,

Houston, TX, USA) was then dissolved in a small amount of ethanol and diluted with

dopamine solution to a final concentration of 5×10-4

g/ml referring to published

studies (Bosetti et al. 2014; Yoon et al. 2007). Collagen membranes purchased by

ZhengHai Biotechnology Co., Ltd. (Yantai, Shandong, China) were cut at 4×3×1 mm

and incubated with 100 μl of calcitriol solution at 4°C overnight. Tris-HCl buffer and

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dopamine solution were used as controls. All steps were performed in the dark.

Scanning electron microscopy (SEM; S-2250N, Hitachi, Tokyo, Japan) was used

to observe the surface morphology of the original collagen membrane,

polydopamine-coated collagen membrane and collagen membrane bearing

polydopamine coating absorbed with calcitriol. For this purpose, samples were

mounted on aluminum stubs and sputter-coated with gold particles to a thickness of

10-15 nm.

Animal mandibular bone defect model and tissue preparation

All animal experiments were conducted in accordance with the Guidelines for

Animal Experimentation of the School and Hospital of Stomatology, Shandong

University. Ninety-six Wistar rats (200-250 g, male) were anesthetized with 10%

chloral hydrate (0.4 ml/100 g body weight). After an extra-oral incision parallel to the

inferior border of the mandible, subcutaneous tissues and masseter muscle were

dissected. Bilateral mandible defects were then made around the first molar with a

fissure bur at low speed with copious saline irrigation. The mandibular wound

window was approximately 5×4×1 mm, with the anterior margin 1 mm distal to the

front of the mandible, and the coronal margin was approximately 1 mm apical of the

crest of the alveolar bone (Fig. 1D). Collagen membranes immersed in different

solutions were then implanted into the defects. Soft tissues were then sutured, and

antibiotics were administered during the first 3 postoperative days.

Four groups were established to treat bone defects (n=6 animals per group per

time point, Table 1): (i) no graft material (control group); (ii) bare collagen membrane

(CM group); (iii) collagen membrane bearing polydopamine coating (DOP/CM

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group); (iv) collagen membrane bearing polydopamine coating absorbed with

calcitriol (CAL/DOP/CM group).

At 1, 2, 4 and 8 weeks post-surgery, animals were anesthetized and fixed with a

transcardiac perfusion of 4% paraformaldehyde in 0.1 M phosphate-buffered saline

(PBS; pH 7.4). Following fixation, mandibles were then demineralized in 10%

EDTA-2Na solution for 4 weeks at 4°C. After dehydration through a graded

ascending ethanol series, the tissues were embedded in paraffin. Serial horizontal

5-µm-thick sections were cut in the apical-to-coronal direction of the tooth.

Histological examination and image analysis

Hematoxylin and eosin staining and Masson’s trichrome stainings were

performed to identify the osteogenic status of the different groups. Stained sections

were observed and digital images were taken with a light microscope (Olympus

BX-53, Tokyo, Japan). With the aid of Image-Pro Plus 6.2 software (Media

Cybernetics, Silver Spring, MD), newly formed bone volume, expressed as a

percentage (area of newly formed bone/area of original wound (defect)*100%), was

measured at an original magnification of ×40. Specifically, bone defect area was

defined from histologically visible cut edge of cortical bone and newly formed bone

was identified by their woven structure.

Three tissue sections were selected from each of three levels throughout the

whole defect (the apical, middle and coronal levels), and three non-overlapping

images of each section were analyzed to obtain the mean value. Ultimately, the newly

formed bone volume of each group was expressed as the average level of the parallel

samples.

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Immunochemical examination for ALP, Runx2, Type I collagen and OPN

Serial 5-µm-thick paraffin sections were used for immunolocalization of ALP,

Runx2, Type I collagen and OPN. Briefly, dewaxed paraffin sections were pretreated

with 0.3% hydrogen peroxidase for 30 min to inhibit endogenous peroxidase activity

prior to treatment with 1% BSA in PBS (1% BSA-PBS) for 20 min. Sections were

then incubated for 2 h at room temperature with: 1) rabbit antiserum against rat

tissue-nonspecific ALP, generated by Oda et al. (1999) at a dilution of 1:100; 2)

mouse anti-Runx2 antibody (MBL, Nagoya, Japan) at a dilution of 1:50; 3) rabbit

anti-collagen I antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:200; or 4)

rabbit anti-OPN antibody (Abcam, Cambridge, MA, USA) at a dilution of 1:50. After

a PBS rinse, sections were incubated with horseradish peroxidase-conjugated

secondary antibodies (DaKo, Glostrup, Denmark) at a dilution of 1:100 for 1 h at

room temperature. The immunoreaction was visualized with diaminobenzidine

(Sigma-Aldrich, St. Louis, MO, USA). Double-staining of ALP and tartrate-resistant

acid phosphatase (TRAP) was performed as previously reported (Li et al. 2013).

Staining was assessed by light microscopy after faintly counterstaining with methyl

green. Primary antibodies were replaced with PBS for negative controls.

Stained sections were imaged with an Olympus BX53 microscope. Specifically,

three tissue sections were selected from each of the three levels (middle, coronal and

apical). Osteoclast numbers were counted, and the mean optical density of ALP and

Runx2 were measured in three randomly selected non-overlapping microscopic fields

from each section by Image-Pro Plus 6.2 software. In brief, regions of interest (ROI)

were manually selected in a color cube based manner.

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Statistical analysis

All values are presented as means ± standard deviation (SD). Statistical analysis

was performed using SPSS software, with differences between groups being assessed

by analysis of variance and considered statistically significant when P<0.05.

Results

Characterization of the calcitriol loaded collagen membrane scaffolds

Using SEM, the surface of the original collagen membrane was observed to be

highly porous with good pore interconnectivity (Fig. 1A). For the

polydopamine-coated collagen membrane, pore number was significantly reduced,

owing to coverage of the polydopamine layer over the surface pores (Fig. 1B). SEM

micrographs of the CAL/DOP/CM group revealed a certain amount of calcitriol, in

spherical shape, distributed on the surface of the collagen membrane (Fig. 1C).

Histological and statistical analysis for bone regeneration

At 1 week post-surgery, new bone nodules emerged. Although fibrous tissue

predominantly occupied the bone defect in the control group, a few areas of new bone

were found scattered above the soft tissue (Fig. 2A). In the CM group, remnants of the

collagen membrane were arranged dispersedly, with some new bone nodules detected

at the bottom of the cavity (Fig. 2B). The DOP/CM group showed a more intact and

regularly assembled collagen membrane, as well as small bone nodules (Fig. 2C). The

CAL/DOP/CM group presented the least amount of collagen membrane residue and

the largest amount of new bone nodules. Furthermore, some regions of the collagen

membrane had been partially replaced by new bone (Fig. 2D).

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At 2 weeks post-surgery, a meshwork of thin bone with large bone marrow

cavities could be identified. An abundance of fibrous tissue remained in the control

group (Fig. 2E). However, the collagen membrane had been mostly degraded in the

CM group and obvious new bone was detected (Fig. 2F). The DOP/CM group was

characterized by considerable collagen residues, with new bone adjacent to the defect

edge (Fig. 2G). The CAL/DOP/CM group showed the most abundant amount of new

bone, occupying the defect up to two-thirds the height of the previous alveolar ridge

(Fig. 2H).

Masson’s trichrome staining was further performed to distinguish between

connective tissue, mineralized and non-mineralized bone. Generally, primary bone

mainly made up of collagen type I is stained into blue while highly mineralized bone

is stained into red. At week 1, control group showed blue-stained fibrous tissue and a

few new bone nodules (red) (Fig. 3A). In the CM and DOP/CM group, the new bone

was mostly blue stained while CAL/DOP/CM group emerged a certain amount of

mineralized bone (red) (Fig. 3B-D). At week 2, primary bone nodules (blue)

distributed dispersedly among fibrous tissue in control group (Fig. 3E). In other three

groups, primary bone (blue) and highly mineralized bone (red) both were observed

(Fig. 3F-H). They staggered mutually forming in an ordered structure especially in

CAL/DOP/CM group (Fig. 3H).

At 4 weeks post-surgery, the new bone featured thicker trabeculae. The control

group showed fibrous healing characteristics, namely fibrous tissue invasion into the

defect with new bone failing to attach to the pre-existing bone (Fig. 4A). In the other

three groups, new bone almost covered the entire bone defect area (Fig. 4B–D). As

shown in Fig. 4, the CM and DOP/CM groups contained more woven bone (Fig.4B

and C) while the CAL/DOP/CM group showed highly mineralized bone matrix (Fig.

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4D).

At 8 weeks post-surgery, new bone was highly mineralized and featured compact

bone. Fibrous tissue was still visible in the control group (Fig.4E). The bone marrow

cavities in the CM and DOP/CM groups were much larger than those in the

CAL/DOP/CM group, which were closer to the cortical bone (Fig. 4F–H).

Masson’s trichrome staining showed consistent results with HE staining at week

4. In control group, abundant fibrous tissue (blue) occupied in the defect area (Fig.

5A). CM group and DOP/CM group showed defects containing more primary bone

(blue) and less mineralized bone (red) (Fig. 5B,C). Conversely, there was mass of

mineralized bone (red) with tiny primary bone (blue) in CAL/DOP/CM group (Fig.

5D). At week 8, control group still showed blue stained fibrous tissue (Fig. 5E).

CAL/DOP/CM group has more mineralized bone (red) and less primary bone (blue)

compared with CM group and DOP/CM group (Fig. 5F-H).

As shown in the schema graph (Fig. 6A), the control group displayed fibrous

healing characteristics, the DOP/CM group was characterized by delayed degradation

of the collagen membrane, while the CAL/DOP/CM group showed accelerated

collagen membrane degradation and bone regeneration. Statistical analysis revealed

that at week 1, newly formed bone volume increased successively in the order of the

control, DOP/CM, CM and CAL/DOP/CM groups. At week 2, newly formed bone

volume in the CAL/DOP/CM group was highest, followed by the CM group. Control

and DOP/CM groups were the lowest with no significant difference between each

other. At weeks 4 and 8, the control group exhibited the least amount of bone, with no

significant difference in bone volume among the other three groups (Fig. 6B).

Immunohistochemical examination and statistical analysis

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At 1 week post-surgery, the control, CM and DOP/CM groups all showed large

populations of TRAP positive osteoclasts adjacent to the bottom of the defects.

However, osteoclasts were barely detected in the CAL/DOP/CM group (Fig. 7A–D).

In contrast, abundant ALP positive osteoblasts were found in the same area in the

CAL/DOP/CM group, with fewer numbers seen in other three groups (Fig. 7A–D).

Consistently, there were significantly more Runx2-positive osteoblasts in the

CAL/DOP/CM group compared with the other three groups (Fig. 7E–H).

Not surprisingly, at 2 weeks post-surgery, the number of osteoclasts decreased in

all groups. However, there remained a faint inhibitory effect when calcitriol was used

(Fig. 7I–L). As expected, Runx2 was highly expressed only in the CAL/DOP/CM

group (Fig. 7M–P) while ALP immunoreactivity was faintly detected in all groups

(Fig. 7I–L).

Statistic analysis further revealed that ALP and Runx2 immunoreactivity both

increased dramatically after calcitriol administration at week 1 (Fig. 8A and B). At

week 2, a significant increase in Runx2 was observed in the CAL/DOP/CM group

with no significant difference in ALP activity among groups (Fig. 8A and B). In

addition, there was reduction in osteoclastogenesis, which could be attributed to

calcitriol administration both at weeks 1 and 2 (Fig. 8C).

At week 4, the control and CAL/DOP/CM groups showed uniform collagen

structure (Fig. 9A and D). Uneven immunostaining was seen in the CM and DOP/CM

groups (Fig. 9B and C). Cement lines showed obvious OPN immunoreactivity in the

CM and DOP/CM groups (Fig. 9F and G). However, in the control and

CAL/DOP/CM groups, OPN was only faintly expressed (Fig. 9E and H). At week 8,

similar results were observed. Newly formed bone was more highly matured in the

control and CAL/DOP/CM groups compared with the other two groups (Fig. 9I–P).

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Discussion

In this study, we investigated the potential role of calcitriol in bone tissue

engineering using a rat mandibular bone defect model. At the early stage of bone

defect repair (weeks 1 and 2), calcitriol exhibited positive effects on bone remodeling

by reducing osteoclastogenesis and promoting osteoblast differentiation, leading to

accelerated bone regeneration. During the subsequent bone maturation phase (weeks 4

and 8), calcitriol regulated collagen maturation and promoted bone mineralization

(Fig. 10).

Tratrate-resistant acid phosphatase (TRAP) , as a glycosylated metalloenzyme, is

highly expressed in osteoclasts and widely used as a specific marker of osteoclasts in

bone (Cole and Walters. 1987; Minkin. 1982). With respect to osteoclastogenesis, our

results that calcitriol inhibit TRAP-positive osteoclasts are consistent with some

previous studies, in which calcitriol inhibited osteoclast formation. Specifically,

calcitriol was reported to suppress the expression of c-fos and nuclear factor of

activated T cells c1 (NFATc1), both of which are key regulators of osteoclast

formation (Sakai et al. 2009; Takasu et al. 2006). However, other studies have shown

that calcitriol strongly induces both receptor activator of NF-B ligand (RANKL) and

macrophage colony-stimulating factor (M-CSF) in vitro and in vivo and induces bone

resorption (Sato et al. 2007; Suda et al. 1999). More interestingly, in

thyroparathyroidectomized rats infused with parathyroid hormone (PTH),

systemically administered calcitriol inhibited PTH-induced bone resorption at a

physiological dose and stimulated bone resorption at a toxic dose (Ueno et al. 2003).

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Taken together, calcitriol may function by both stimulating and inhibiting bone

resorption depending on the dose and methods of administration. Furthermore,

calcitriol has opposing effects in osteoblast differentiation, which may be due to

differences in animal species and reagents (Hicok et al. 1998; Li et al. 2008; Lohmann

et al. 2000). For example, Li et al. evaluated the in vitro effects of 1,25(OH)2D3 on

osteoblasts from three different species and found that in rat cells, the active vitamin

D increased cell proliferation, inhibited cell apoptosis and increased alkaline

phosphatase (ALP) activity. In mouse cells, however, it initiated cell apoptosis and

inhibited ALP activity. In human cells, although cell proliferation and apoptosis was

while ALP activity was enhanced (Li et al. 2008). In the current study, calcitriol

enhanced both ALP- and Runx2-immunoreactivity at week 1 and promoted Runx2

expression at week 2. These findings further confirm that ALP and Runx2 exhibit

differential expression during osteoblast differentiation (Reichert et al. 2013).

Enhanced bone regeneration observed at weeks 1 and 2 may be attributed to

inhibited bone resorption and induced bone formation. The detailed mechanisms of

calcitriol in bone formation is still the ongoing controversy. Phex, a marker of the

mature osteoblasts, and Runx2, a key regulator of bone formation in vivo, were

regulated by calcitriol (Drissi et al. 2002; Ecarot et al. 1999). A recent study showed

that calcitriol binding to VDR regulates BMP2 gene expression by binding to the

BMP2 promoter region and inducing DNA methylation and histone modification (Fu

et al. 2013). Another noteworthy problem is that during normal bone remodeling,

bone deposition usually depends on the accurate coordination between osteoclasts and

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osteoblasts (Wright et al. 2005). The current study identified a discrepancy between

inhibition of osteoclastogenesis and promotion of osteogenic differentiation. This

phenomenon can perhaps be partly interpreted by our previous findings (Saito et al.

2013), in which eldecalcitol, a second-generation vitamin D analog, induced focal

bone formation in absence of osteoclasts, referred to as ‘bone mini modeling’.

Another possible explanation for this discrepancy is that osteoclastogenesis occurred

at earlier stages which escaped our detection. The specific mechanism for this remains

to be elucidated.

A previous study demonstrated that calcitriol directly regulates collagen

cross-linking in an osteoblast-like cell line (MC3T3-E1), likely by up-regulating gene

expression of specific LH and LOX enzymes (Nagaoka et al. 2008). This partially

explains our findings, that calcitriol induced collagen maturation and promoted bone

mineralization at weeks 4 and 8. The mechanism of calcitriol in promoting bone

mineralization is complex. In addition to its systemic effects on mineral ion

homeostasis, calcitriol has been proposed to have a direct effect on mineralization

through the VDR on skeletal cells (Chen et al. 2013). Furthermore, calcitriol can

stimulate mineralization of human osteoblast cultures (Miyahara et al. 2002; van Driel

et al. 2006) and modulate expression of various mineralization-related genes, such as

OCN (BGLAP) and OPN (SPP1) (Barthel et al. 2007; Shen and Christakos 2005).

However, high-dose 1,25(OH)2D3 is reported to inhibit osteoblast mineralization in

vitro (Yamaguchi and Weitzmann 2012).

The physiological effect of calcitriol is closely related to its dosage. We chose

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the dose at 5×10-4

g/ml referring to published studies which decare using calcitriol at

10-4

g/ml level with scaffolds showed good potential to implement osteoinduction and

osteogenesis in vivo (Bosetti et al. 2014; Yoon et al. 2007). In our present study,

calcitriol showed more significant effect on osteogenesis at week-1 and week-2 than

week-4 and week-8 owing to dose attenution. Therefore, it needs more study to find

more appropriate dose of calcitriol.

In the current study, another interesting phenomenon was that polydopamine

delayed the degradation of collagen membrane. As described in a previously

published paper, polydopamine coating is stable and not easily degraded. It acts as an

impermeable cap, preventing the release of Ca2+

and PO43-

from BCP scaffolds and

slowing their degradation (Jia et al. 2013). It is therefore not surprising that

polydopamine formed a stable layer adherent on the surface of the collagen membrane,

protecting it from degradation. However, the exact mechanism by which calcitriol

accelerates degradation of the collagen membrane requires further study.

Calcitriol is liposoluble and it could be very hard to immoblize it on scaffolds

without modification method. That is the reason why we use poly-dopamine coating

instead of using calcitriol only. In our present study, CAL/DOP/CM group exhibited

positive effects on bone remodeling and mineralization while DOP/CM group showed

no silimar action. It gives a strong evidence that calcitriol only can promote

osteogenesis. From another aspect, polydopamine delayed the degradation of collagen

membrane while calcitriol accelerated it completely offseting the effect of

polydopamine. Therefore, we could have observed promoted osteogenesis and more

obviously accelerated degradation of collagen membrane if there was CAL only group.

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The follow-up study to confirm this phenomenon depends on some new technologies.

For GBR, a barrier film is often placed covering the bone defect to create a

relatively closed environment and prevent fibrous tissue invading into the bone defect.

Based on this theory, it is not difficult to understand that the control group showed

fibrous healing characteristics in our study. In addition, the collagen carrier for

calcitriol was mostly resorbed by 2 weeks, but further studies are necessary to identify

materials that can provide longer-term scaffolding, to support continuing bone

formation in a mechanically challenging environment (Khan et al. 2012).

Conclusions

Our results suggest that local administration of calcitriol accelerates bone

formation and promotes bone maturation, thus providing important experimental data

for the favorable use of calcitriol for bone regeneration. However, considering the

bidirectional actions of calcitriol, more effort needs to be made towards finding the

best characteristics of calcitriol for bone tissue engineering purposes.

Acknowledgements

This study was partially supported by the National Nature Science Foundation of

China (grant No. 81271965; 81470719; 81311140173) and Specialized Research

Fund for the Doctoral Program of Higher Education (grant No. 20120131110073) to

Li M.

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Figure Captions

Fig. 1 SEM micrographs and diagram representing mandibular bone defect

SEM micrographs of the original collagen membrane (A), polydopamine-modified

membrane (B) and calcitriol-polydopamine composite membrane (C). The diagram

represents the surgical window in the buccal surface of the first molar (D). The

surface of the original collagen membrane was highly porous with good pore

interconnectivity (A). The polydopamine-coated collagen membrane displayed

coverage of the polydopamine layer over the membrane surface (asterisk, B).

Calcitriol granules were observed distributed on the surface of the scaffolds (white

arrows, C). Bar, A–C: 1μm

Fig. 2 Hematoxylin and eosin staining of bone defects at weeks 1 and 2

Low-magnification images of bone defects at weeks 1 (A-D) and 2 (E-H). In the

control group, fibrous tissue occupied the bone defect with a small number of new

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bone nodules (A). In the CM group, remnants of the collagen membrane were

arranged dispersedly and some new bone nodules were detected at the bottom of the

cavity (B). More intact and regularly assembled collagen membrane, as well as small

bone nodules, were seen in the DOP/CM group (C). The CAL/DOP/CM group

showed the least collagen membrane residue and largest amount of new bone (D). At

week 2, there was still much fibrous tissue in the control group (E). The collagen

membrane was degraded completely in the CM group and obvious new bone was

detected (F). The DOP/CM group was characterized by considerable collagen residue

while new bone was limited adjacent to the defect edge (G). The CAL/DOP/CM

group showed the most amount of new bone, almost filling the entire bone defect (H).

CB: cortical bone; NB: newly formed bone; M: collagen membrane; F: fibrous tissue.

Bar, A–H: 250 μm.

Fig. 3 Masson’s trichrome staining of bone defects at weeks 1 and 2

Overview images of bone defects at weeks 1 (A-D) and 2 (E-H) showed by Masson’s

trichrome staining. At week 1, control group showed blue-stained fibrous tissue and a

few new bone nodules (red) (A). In the CM and DOP/CM group, the new bone was

mostly blue stained while CAL/DOP/CM group emerged a certain amount of

mineralized bone (red) (B-D). At week 2, primary bone nodules (blue) distributed

dispersedly among fibrous tissue in control group (E). In other three groups, primary

bone (blue) and highly mineralized bone (red) both were observed (F-H). They

staggered mutually forming in an ordered structure especially in CAL/DOP/CM group

(H). CB: cortical bone; NB: newly formed bone; M: collagen membrane; F: fibrous

tissue. Bar, A–H: 250 μm.

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Fig. 4 Hematoxylin and eosin staining of bone defects at weeks 4 and 8

Low-magnification images of bone defects at weeks 4 (A-D) and 8 (E-H). At week 4,

the control group showed fibrous healing characteristics, with new bone failing to

attach to the pre-existing bone because of invading fibrous tissue (A). In the other

three groups, new bone almost covered the entire bone defect area: the CM and

DOP/CM groups presented woven bone, while the CAL/DOP/CM group showed

highly mineralized bone (B-D). At week 8, new bone was highly mineralized and

featured compact bone. Fibrous tissue was still visible in the bone defect of the

control group (E). The bone marrow cavities were much larger in the CM and

DOP/CM groups than those in CAL/DOP/CM group, which was closer to the cortical

bone (F-H). CB: cortical bone; NB: newly formed bone; F: fibrous tissue. Bar, A–H:

250 μm.

Fig. 5 Masson’s trichrome staining of bone defects at weeks 4 and 8

Overview images of bone defects at weeks 4 (A-D) and 8 (E-H). In control group,

abundant fibrous tissue (blue) occupied in the defect area (A). CM group and

DOP/CM group showed defects containing more primary bone (blue) and less

mineralized bone (red) (B,C). Conversely, there was mass of mineralized bone (red)

with tiny primary bone (blue) in CAL/DOP/CM group (D). At week 8, control group

still showed blue stained fibrous tissue (E). CAL/DOP/CM group has more

mineralized bone (red) and less primary bone (blue) compared with CM group and

DOP/CM group (F-H).

Fig. 6 Schema graph of different groups and statistical analysis

The schema graph illustrates the results of the histological analysis (A). Statistical

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analysis for newly formed bone volume (B). The control group showed fibrous

healing characteristics, the DOP/CM group was filled by collagen membrane, and the

CAL/DOP/CM group featured less collagen membrane and more new bone (A). At

week 1, following the sequence of less to more bone volume, the order was control,

DOP/CM, CM and CAL/DOP/CM groups. At week 2, the newly formed bone volume

in the CAL/DOP/CM group was the highest, followed by the CM group. Control and

DOP/CM groups were the lowest, with no significant differences between each other.

At week 4 and 8, the control group exhibited the least amount of bone while there was

no significant difference among the other three groups (B). (n=6; * P< 0.05; ** P<

0.001), Bars indicate ±SD.

Fig. 7 Effect of calcitriol on osteoblasts and osteoclasts at the early stage of bone

repair

Double staining for ALP (brown) and TRAP (red) in bone defect at weeks 1 (A-D)

and 2 (I-L). Immunohistochemistry for Runx2 (brown) in the same visual field at

weeks 1 (E-H) and 2 (M-P). At week 1, the control, CM and DOP/CM groups all

showed large populations of TRAP positive osteoclasts adjacent to the bottom of the

defects, which were barely detected in the CAL/DOP/CM group (A-D). In contrast,

abundant ALP positive osteoblasts (asterisk) were found in the same area in the

CAL/DOP/CM group, with few observed in other three groups (A-D). Consistently,

there were significantly more Runx2 positive cells (asterisk) in the CAL/DOP/CM

group (H). At week 2, the number of osteoclasts decreased in all groups, but a visible

inhibitory effect remained when calcitriol was used (L). Runx2 was highly expressed

(asterisk) only in the CAL/DOP/CM group (P) while ALP immunoreactivity was

faintly detected in all groups (I-L). oc: osteoclast. Bars, A–H: 25 μm.

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Fig. 8 Statistical analysis for ALP and RUNX2 activity and TRAP positive

osteoclast number

Statistical analysis for ALP activity (A), Runx2 activity (B) and TRAP positive cell

number (C). ALP and Runx2 immunoreactivity both increased dramatically after

calcitriol administration at week 1 (A, B). At week 2, Runx2 still showed a significant

increase in CAL/DOP/CM group while there was no significant difference in ALP

activity (A, B). In addition, osteoclast number was significantly reduced in the group

of calcitriol administration both at weeks 1 and 2 (C). (n=6; ** P< 0.001). Bars

indicate ± SD.

Fig. 9 Effect of calcitriol on bone maturation and mineralization

The immunohistochemistry for Type I collagen (brown) at weeks 4 (A–D) and 8 (I–L).

Immunohistochemistry for OPN (brown) in the same visual field at weeks 4 (E–H)

and 8 (M–P). At week 4, the control and CAL/DOP/CM groups showed uniform

collagen structure (asterisk) (A, D). Uneven immunostaining was seen in the CM and

DOP/CM groups (B, C). Cement lines showed obvious OPN immunoreactivity in the

CM and DOP/CM groups (black arrows, F, G). In the control and CAL/DOP/CM

groups, OPN was only faintly expressed (E, H). At week 8, similar results were

observed, newly formed bone in the control and CAL/DOP/CM groups was more

highly matured than in the other two groups (I–P). Bar, A–H: 25 μm.

Fig. 10 Hypothetical scheme illustrating the influence of calcitriol on restoration

of mandibular bone defects

During initial bone remodeling, calcitriol inhibits osteoclastogenesis and promotes

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osteoblast differentiation. During subsequent bone maturation, calcitriol regulates

collagen maturation and promotes bone mineralization.

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Table 1

Experimental groups

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Highlights (for review)

More information on collagen material was added in the revised manuscript.

Masson-Goldner trichrome stain was performed for histomorphometry.

More specific information on calcitriol was supplemented in Discussion section.

The MOD of ALP and Runx2 was explained in more detail.

The inhibition of osteoclastogenesis was described more accurately in the second

paragraph of the discussion.